Novel plant agglutinin gene

ABSTRACT

The present invention provides the cloning of intron-containing  Amaranthus caudatus  agglutinin (ACA) gene from  Amaranthus caudatus  and the coding region gene sequence encoding the ACA protein. The said gene sequence of the ACA gene coding region was ligated with highly efficient and stable microbial expression vectors to produce the ACA protein using microbe largely. High expression of the ACA gene can make the transgenic plants have antiaphid activity and improve the nutrition quality thereof. The ACA gene has potential application value in plant anti-insect gene engineering and quality improvement.

FIELD OF THE INVENTION

[0001] This invention relates to the field of plant gene engineering for insect-resistance and biochemistry. Particularly, it relates to a newly cloned Amaranthus caudatus agglutinin (ACA) gene and the recombinant plasmids for conducting the expression of ACA gene in plants. Transgenic plants having aphid-resistance can be obtained from the transformation of plants with the said recombinant plasmids. Meanwhile, ACA protein could be produced using microbes harboring a high efficient and stable microbe expression vector for ACA gene.

BACKGROUND OF THE INVENTION

[0002]Amaranthus caudatus agglutinin (ACA) is a kind of phytoagglutinin (plant lectin) present in the seeds of Amaranthus caudatus having high nutritive value originated in South America continent [Vietmeyer, N. et al (1986), Science 232:1379-1384] and is mainly as a storage protein providing nutrition during the seed germination. ACA is abundantly synthesized in seeds during the formation of endosperm, but little in vegetative organs. The amino acid sequence of ACA protein has been reported in GenBank (Accession No. g2781234). Rahbe Y. et al carried out in vitro feeding pea aphids (Acyrthosiphon pisum) with different purified plant lectin proteins and the results showed that ACA gave the very high inhibition or toxic effect for tested aphids [Rahbe Y, Sauvion N, Febvay G et al 1995, Entomol Exp Appl, 76:143-155]. It is reported that this agglutinin plays an important role in specific recognition of tumor cells, histochemical identification and early diagnosis of tumors [Boland, C. et al, (1991), Cancer Res. 51:657-665]. As the function of plant agglutinins being further elucidated and potential application values thereof in medicine and biology being understood, it appears necessary to clone the ACA gene for studying and applying the agglutinin. Though the aphid-resistant activity of ACA has been revealed in the insect-resistant test in vitro as described, this activity has not been verified in transgenic plants expressing ACA gene. Moreover, ACA protein as a protein being rich in essential amino acid residues may play an important role in improving quality of crop. High level expression of ACA protein obtained in vitro conveniently could be a sufficient supply of this protein for identification of some tumor diseases.

BRIEF DESCRIPTIONS OF THE INVENTION

[0003] Based on the above situation, the purpose of this invention is to provide a novel structural gene of ACA protein, its recombinant plasmid and recombinant plasmid expressed in microbe and plants.

[0004] In order to obtain the structural gene of ACA, the structural gene of ACA was first obtained by polymerase chain reaction (PCR) using total DNA from A. caudatus as template. The gene consists of 2628 bp including an intron of 1716 bp and two exons of 212 bp and 700 bp respectively. High sequence homology between the amino acid sequence deduced from the nucleotide sequence thereof and the reported amino acid sequence of ACA protein reported by transue et al[Nature Structural Biology,1997,4(10):779-783] exists. A recombinant plasmid which can drive the ACA gene constitutively expressed in plants was constructed. The advantage thereof is that the recombinant plasmid can express the ACA protein in all tissues of the resulted transgenic plants. For expression of the ACA gene in particular tissue, a tissue-specific promoter could be used to make the ACA protein specifically be synthesized in the target tissue of transgenic plants. This type of expression of ACA protein could make the transgenic plants resistant to aphid efficiently. At the same time, since ACA protein is rich in essential amino acids, its high expression in transgenic plants could improve the nutritive value of the plants. An efficient and stable expression vector is constructed and abundant ACA protein could be synthesized in recombinant microbes, the protein could have certain application in tumor-detection.

[0005] The ACA gene is firstly cloned by this invention, this makes it possible to produce ACA in large quantity and to apply this agglutinin in agriculture and medicine.

[0006] In order to achieve the above purposes, the particular procedures leading to this invention were as follows: A pair of PCR primers were designed and synthesized according to the cDNA sequence of AmA1 protein of Amaranthus hypochondriacus [Anjana R et al. (1992). PNAS, Vol(89):11774-11778], which is highly homologous in amino acid sequence with ACA protein. ACA structural gene was amplified by PCR in the presence of genomic DNA extracted from seeds or leaves of A. caudatus and cloned. The purified PCR product was ligated with pUC18 vector and then transformed into E. coli DH5α to obtain the recombinant plasmid pACA containing ACA structural gene and E. coli DH5α transformant thereof. The transformant (pACA/DH5α) named pACAg/DH5α was deposited in the China General Microbiological Culture Collection Center with accession No. CGMCC NO.0438 A pair of PCR primers were designed according to the sequence of the recombinant plasmid pACA which was used as a template, the DNA fragment in the coding region of cDNA of ACA was amplified by reverse PCR, and then transformed into E. coli DH5α after self-ligation to obtain the recombinant plasmid pACAc and the E. coli DH5α transformant thereof. DNA fragments of the cDNA and ACA structural gene were isolated from recombinant plasmid pACAc and pACA, and ligated with plant constitutive expression vector pBin438 respectively and then transformed into E. coli DH5α to obtain recombinant plasmids pBACAc and pBACA that can express constitutively in plant and E. coli DH5α transformant. Agrobacterium tumefaciens LBA4404 was transformed with recombinant plasmid pBACAc and PBACA extracted from the above transformants to obtain A. tumefacieus transformants pBACAc/LBA4404 and pBACA/LBA4404 respectively. At the same time, the DNA fragment of the ACA structural gene was isolated from the recombinant plasmid pACAc and ligated with E. coli expression vector pET30a(+), then transformed into E. coli BL21(DE3) to obtain a recombinant plasmid pEACAc that can express in E. coli and E. coli transformant pEACAc/BL21 (DE3). The E. coli transformant pEACAc/BL21(DE3) can express a specific ACA protein upon induction.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The attached drawings are presented to further illustrate this invention in detail.

[0008]FIG. 1: Construction of a recombinant plasmid pACA of the ACA gene (Amp: ampicillin resistance)

[0009]FIG. 2: Total length nucleotide sequence of the ACA gene, letters in lower case represent the nucleotide sequence of the intron.

[0010]FIG. 3: Sketch map of the ACA gene structure, numbers on the map indicate the size of corresponding region in base pairs.

[0011]FIG. 4: Homologous comparison of amino acid sequence of the ACA protein deduced from the nucleotide sequence of ACA gene with the amino acid sequence of the ACA protein reported (ACA.PRO: amino acid sequence of the ACA protein; ACAc.PRO: Amino acid sequence deduced from the cloned ACA gene of the invention; amino acids in boxes indicate the differences between the two sequences)

[0012]FIG. 5: Construction of plant expression vector PBACA for ACA gene (NPTII: Neomycin phosphotransferase gene; 35S: CaMV 35S promoter with double enhancer sequences; NOS: Transcription terminator sequence of nopaline synthase gene; LB: T-DNA left border of Ti plasmid; RB: T-DNA right border sequence of Ti plasmid)

[0013]FIG. 6: Construction of plant expression vector pBACAc for ACA gene coding region (Kl: Kienow DNA polymerase)

[0014]FIG. 7: SDS-PAGE pattern of E. coli expression products of the ACA protein [M: Protein molecular weight standard; 1: pEACAc/BL21(DE3) 2: pET30a/BL21(DE3); Arrowhead points the position of the expressed target protein (ACA)]

DETAILED DESCRIPTION OF THE PREFERABLE EXAMPLES

[0015] Further detailed description of this invention is presented via particular examples and the attached figures.

EXAMPLES

[0016] 1. Isolation of Total DNA from Amaranthus Caudatus Plants

[0017] Seeds of Amaranthus caudatus were kindly provided by Regional Plant Introduction Station of USDA, IOWA State University. The procedure of DNA isolation was referred to Paterson A H, Brubaker C L, Wendel J F et al [Plant Mol. Biol. Reporter, 1993, 11(2): 122-127] with some modifications. Two hundreds mg of leaves from Amaranthus caudatus plants or seeds that is maturing were ground into powder in liquid nitrogen and 1 ml of DNA extraction buffer [0.35 M glucose, 0.1 M Tris-HCl (PH8.0), 0.005 M Na2-EDTA (PH8.0), 2% (W/V) polyvinyl-pyrolidone (PVP40), 0.1% (W/V) diethylithiocabarmic acid (DIECA), 0.2% (W/V) mercaptoethanol, 0.5% Triton-X100] prechilled with ice was added. After mixed thoroughly, the suspension was centrifuged at 4° C., 2700×g for 10 mins to remove the supernatant. To the resulted pellet, 500 μl of nuclear lysis buffer [0.1 M Tris-HCl, PH8.0, 1.4 M NaCl, 0.02 M Na2-EDTA (PH8.0), 2% (W/V) CTAB (hexadecy1triammonium bromide), 2% (W/V) PVP, 0.1% (W/V) DIECA, 0.2% (W/V) mercaptoethanol, 0.5% Triton-X100] was added for re-suspension of the pellet at 65 ° C. for 30 mins. The resuspension was extracted with equal volume of chloroform-isoamyl alcohol (24:1) and to the resulted supernatant (the up-phase) after centrifugation at 10000 rpm, 0.6 vol of pre-cold isopropanol was added to precipitate DNA. After centrifugation at 12000 rpm, the precipitate was washed with 70% ethanol, dried at room temperature and dissolved in 50 μl sterilized water and stored at −20° C.

[0018] 2. PCR Amplification of the ACA Gene

[0019] Twenty μl of a PCR system consisted of 1 μl of about 50 ng total DNA from Amaranthus caudatus, 2 μl 10×PCR buffer, 2 μl dNTP (the final concentration for each NTP is 10 mmol/L), 2 μl each of the two primers (final concentration is 10 pmol/μl), Taq Plus I (2.5 Units), the rest volume was made up with deionized water. The reaction system was covered with proper amount of mineral oil to prevent evaporation. 5′ primer sequence: 5′-GGA AGA TCT ACC ATG GCG GGA TTA CCA GTG-3′ 3′ primer sequence: 5′-AGC GTC GAC TTA GTT GTT GGA TCC CAA TTC-3′

[0020] The reaction condition is: pre-denaturing at 94° C. for 3 mins, and then 94° C. 1 min, 49 ° C. for 1 min, 72° C. 1 min 30 sec, with 30 cycles, an elongation of 10 mins at 72° C. was preformed after the 30 cycles reaction. The PCR products were separated by electrophoresis and two DNA fragments of 2.5 kb and 0.9 kb were isolated. Recovery of the two fragments was performed using the DNA recovery kit available from Shanghai Huashun Ltd and dissolved in 30 μl sterilized water.

[0021] 3. Cloning and Sequencing of the ACA Gene

[0022] Methods for DNA clone and sequencing referred to ((Molecular Cloning, A Laboratory Manual)) edited by Sambrook J. et al., Cold Spring Harbor Laboratory Press, 1993. 10 μl of the recovered PCR product was ligated with 1 μl of pUC18-T vector in 20 μl of ligation system comprising 2 μl of 10×ligation buffer, 10 μl of PCR product, 1 μl of vector, 1 μl of T4 DNA ligase and 6 μl of water. The ligation reaction was performed at 14° C.˜16° C. for 12 hours. E. coli DH5α competent cells was transformed with 8 μl of the ligation mixture and then spreaded onto the LB solid medium containing 50 mg/L Amp (ampicillin). The positive colonies were selected using blue-white colony screening method. The plasmids of selected positive colonies were extracted and further identified by restriction enzyme digestion and sequencing analysis using Pharmacia T7 DNA Sequencing Kit. The results confirmed the insertion of the 2.6 kb ACA gene. The construction process of the recombinant plasmid is shown in FIG. 1, named as pACA. The complete nucleotide sequence of the ACA gene was analyzed on an ABI 377 DNA sequencer and the result is shown in FIG. 2. The result from sequence analysis indicate that the ACA gene is consisted of 2628 bp, containing an intron of 1716 bp and two exons of 212 bp and 700 bp respectively. The structure of the ACA gene is shown in claim 1 and diagramed in FIG. 3.

[0023] 4. Coding Sequence of the ACA Gene Obtained by Reverse PCR

[0024] Based on the sequence of the ACA gene, two primers complemented with the exons adjacent to the intron respectively were designed and synthesized. The sequences of the two primers were as followes: 5′ end sequence: 5′-GTG GTC TCC CAA TCA TTA TTG-3′ 3′ end sequence: 5′-CTA ACC AAA TAT TTG TTA GTG-3′

[0025] Reverse PCR was performed in a total volume of 20 μl containing 1 μl dNTP (10 mmol/L each); 1 μl of each primer (25 pmol/μl); 1 μl of the diluted pACA solution as template (pACA extracted from E. coli transformant by alkaline lysis method was diluted 500 fold); 2 μl of 10×PCR buffer, 0.4 μl of pfu Taq DNA polymerase (5 uint/μl), the final volume was made up with sterilized water. The reaction condition is: pre-denaturing at 94° C. for 3 mins, and then 94° C. 50 sec, 58° C. for 50 sec, 72° C. 2 mins 30 sec, with 30 cycles, an elongation of 10 mins at 72° C. was preformed after the 30 cycles reaction. The PCR product showed a 3.5 kb DNA in size on Agarose gel electrophoresis. The PCR product was purified and then phosphorylated by T4 polynucleotide kinase. After precipitated with ethanol, the DNA pellet was dissolved in 20 μl sterilized water.

[0026] 5. Cloning and Sequencing of the Coding Region of the ACA Gene

[0027] 2 μl of 10×ligation buffer, 1 μl T4-DNA ligase (1 Unit/μl) and 7 μl of sterilized water was added to 10 μl of the above purified product, the final volume was 20 μl. The reaction mixture was incubated at 14˜16° C. for 12 hours. Competent cells of E. coli DH5a was transformed with 8 μl of the ligation mixture and spreaded onto the LB solid medium containing 50 mg/L Amp. Plasmids in the selected transformants were isolated using alkaline lysis method and a 900 bp fragment was produced after digestion with Nco I and Sal I as expected. The recombinant plasmid is designated as pACAc. The coding sequence in pACAc was verified by sequence analysis on an ABI 377 DNA sequencer. The coding sequence of ACA gene in pACAc is shows in FIG. 2 (represented by capital letters). Comparison of the amino acid sequence encoded by ACA gene with that of the published ACA sequence is shown in FIG. 4.

[0028] 6. Construction of Plant Expression Vector for ACA Gene Coding Sequence and Preparation of Recombinant Agrobacterium Tumefacieus.

[0029] Plasmid pACAc was digested by Bgl II and Sal I. The fragment of the ACA structural gene was recovered and then ligated with the fragment of the vector pBin438 digested by BamH I and Sal I [Li T-Y, et al, Science in China (B series), (1994) 24: 276-282] to construct the recombinant plasmid pBACAc, as outlined in FIG. 5. After transformation of E. coli competent cells with the ligation mixture, the cells were spreaded onto the LB solid medium containing 50 mg/L Kan(kanamycin). The recombinant plasmid was identified by restriction enzyme digestion and PCR method. Purified pBACAc using alkaline lysis method was transformed into competent cells of Agrobacterium tumefacieus LBA4404, the cells were spreaded onto the YEP solid medium containing 50 μg/μl Kan, 50 μg/μl Rif(rifampin), 50 μg/μl Str(streptomycin). The selected single colony was analyzed by restriction enzyme digestion and PCR detection to obtain the tranformant of Agrobacterium tumefacieus LBA4404 containing pBACAc.

[0030] 7. Construction of Plant Expression Vector Containing the ACA Gene

[0031] The ACA gene fragment from pACA was digested by Nco I, filled in the cohesive ends with klenow DNA polymerase and then digested by Sal I to obtain a 2.6 kb of gene fragment. The isolated fragments were ligated with vector pBin438 fragment digested by BamH I and then filled in with klenow followed by digested with Sal I to form recombinant plasmid pBACA. The ligation mixture was transformed into competent cells of E. coli DH5α and the recombinant plasmids in the transformants selected were identified by restriction enzyme digestion and PCR detection. The preparation of Agrobacterium tumefacieus was as the same with that of described in above 6. Besides CaMV 35S promoter, tissue-specific promoters or other kind of promoters could be used to construct a plant expression vector with the ACAc or ACA gene. The construction process was shown in FIG. 6.

[0032] 8. Construction of a Expression Vector for ACAC Gene and its Expression in E. coli.

[0033] The plasmid pACAc was digested by Nco I/Sal I. About 0.9 kb fragment was recover and then ligated with a expression vector pET30a (+) digested by the same enzymes. The recombinant plasmid named as pEACAc was used to transform E. coli BL21 (DE3) cells. Induced expression of the ACA gene was achieved based on the procedure provided by Qiagene Company[A handbook for high-level expression and purification of 6×His-Taged protein]. A single colony of pEACAc transformants was inoculated into liquid LB medium containing 50 μg/μl kamamycin and cultured at 37° C. with shaking at 250 rpm. When the bacteria grow to OD=0.2, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mmol/L. The culture was shaked continually in the same condition for 3h. Total protein of the bacterial cells was analyzed by SDS-PAGE as described by Laemmli UK. (Nature 227:680-685, 1970) using pre-stained protein marker from BioLab as protein MW standard. Results from SDS-PAGE showed that a 36 kD specific protein was produced in pEACAc transformant (FIG. 7, lane 1) while this protein is absent in E. coli BL 21 (DE3) cells (FIG. 7, Lane 2), indicating that the ACA protein was expressed in E. coli. The expression level of the ACA protein is estimated to be 35% of total bacterial proteins based on the calculation of gel-scanning. SDS-PAGE pattern was shown in FIG. 7.

[0034] Notes:

[0035] LB medium: 5 g/L NaCl; 10 g/L Trypton; 5 g/L Yeast extract; PH7.0; 12 g/L Agar; Autoclaved at 15 pound for 30 mins.

[0036] YEP medium: 5 g/L NaCl; 10 g/L Trypton; 10 g/L Yeast extract; PH7.0; 12 g/L Agar, Autoclaved at 15 pound for 30 mins.

[0037]

1 4 1 2628 DNA Amaranthus caudatus 1 atggcgggat taccagtgat tatgtgccta aaatcaaata acaaccagaa gtacttaaga 60 tatcaaagtg ataatattca acaatatggt cttcttcaat tttcagctga taagatttta 120 gatccattag ctcaatttga agtcgaacct tccaagactt atgatggtct tgttcacatc 180 aaatctcgct acactaacaa atatttggtt aggtacgttt tttttcgcgt ttatacttcc 240 tccgtttcaa tatagtcgca atatatcgaa aataaacact attcacttat ctcctttaat 300 ttgtgattga taagtaattt ataagttaaa acatagtcaa ctgagatctt gtttgattca 360 tctcgacgta aagattatta atatcaaatt tttataattt tatattatac ataattgtag 420 ttattaatga ttgaattagt acattagact gcgtgaaaaa ggcatatgtt gtaagtattt 480 tggaacgaag gtagtgttat atttcttctg ttagtctatt taaaatttgc gatatttaaa 540 ttaaatattt aagatttttt tgagttacat gtttcaaatt ctggagtgag tacgtacttt 600 aagtatactt ttgtactagc taccatttga ttgacataat taataaatgg gaaattccac 660 atggtagcat ctagttttga tgaaatgcca gtggtgacac ttttttaaga aaattccaca 720 tgctagcatt cagtttatac actatctaat tgccgtagca ttttccgtta agttcttgtt 780 aaattccact aaatttatca ttttgtaagt ttttttccac atggtagtat ccagtttttg 840 ttaatttatc attttaactc ttaattcacc attttaatgt ttaattcact atttaattca 900 ttaacgctca taagtgttag atatttttat tgaaaaatta taagaaacta tctaatcggg 960 tcgaaacaag aacttattcg cctatttttc gatacgtaaa ccggaaaaga tatttggcgt 1020 catttttacc tatcataacg atttttttca ataaacggac gttgcaatta cccttatggg 1080 ataactcttt cgaaaatccg gtcgaaacaa gtacttattc gcctattttc cgatagatgt 1140 gttcaagaaa atgggacaac taaatagatt cagagggagt atttcggagt aattatgttg 1200 acaaaattat acttgaacta atagctacgg tgatgcttgt tcaatttcac tgattttgta 1260 caatcataaa gtttcttgat cacatttcca aaaaaatgaa agttaagtgg caaaaaatat 1320 gtgaatataa aatttgacaa gtctaattta aaattttcac taattttttt taataaaacg 1380 aaatacaaca taatatattt ttattgagat atattttgtg aaatttaatt taaatgtcac 1440 cactataaat ttgtaatggt acatatagtt gatttagtta catttattag accttctagc 1500 tgcataatta aaatcatact aaagcttttt cctgagatag aatctaatga tatttctcat 1560 atgccggcat gcaaccaaat aaagtgttac tatataaatt actaaagtag cgacattttt 1620 taatgttgca aaaagaaaat tttgtttagt gacggtctta tagtgtgact atctctattg 1680 ggcagacata tgtacattta gtatgaaatg atcacttata attttaaagt aatacaatta 1740 cagagtgact tgtttacaat gaatttgtgt ttttgtccag caagtctttt aatgagagac 1800 gtctcttaga tctaatccat tagagtttaa ttcttactct tattctatat ttttctttat 1860 gggtcaccca attagagttg gtatctcaaa gagaccgtct ctcacaagaa tttgcgtttt 1920 tgtcttaggt ggtctcccaa tcattattgg attacagcat cagccaatga accagatgaa 1980 aataaaagca attgggcatg cacattattc aaaccacttt acgtagaaga aggtaacatg 2040 aaaaaggttc gacttttgca cgtccaatta ggtcattata cacagaatta taccgttggt 2100 gggtccttcg tatcatactt atttgccgaa tcaagtcaaa ttgataccgg ctctaaagac 2160 gtattccatg tcatagattg gaaatcaatc tttcaatttc ccaaaagata tgtcacattt 2220 aaaggaaata atggaaaata tttaggggtt atcacaatta atcaacttcc atgtctacaa 2280 tttgggtatg ataatcttaa tgatccaaag gtggctcatg aaatgtttgt cacttctaat 2340 ggtactattt gcattaaatc cacttatatg aacaagtttt ggagactctc tacggatgat 2400 tggatattag tcgatgggaa tgatcctcgc gaaactaatg aagctgcagc gttgtttagg 2460 tcagatgtgc atgattttaa tgtgatttcg cttttgaaca tgcaaaaaac ttggtttatt 2520 aagagattta cgagtggtaa gcctgggttt ataaattgta tgaatgcagc tactcaaaat 2580 gttgatgaaa ctgctatttt agagataata gaattgggat ccaacaac 2628 2 212 DNA Amaranthus caudatus Exon 1 of Amaranthus caudatus agglutinin structure gene (1)..(212) 2 atggcgggat taccagtgat tatgtgccta aaatcaaata acaaccagaa gtacttaaga 60 tatcaaagtg ataatattca acaatatggt cttcttcaat tttcagctga taagatttta 120 gatccattag ctcaatttga agtcgaacct tccaagactt atgatggtct tgttcacatc 180 aaatctcgct acactaacaa atatttggtt ag 212 3 700 DNA Amaranthus caudatus Exon 2 of Amaranthus caudatus agglutinin structure gene (1)..(700) 3 gtggtctccc aatcattatt ggattacagc atcagccaat gaaccagatg aaaataaaag 60 caattgggca tgcacattat tcaaaccact ttacgtagaa gaaggtaaca tgaaaaaggt 120 tcgacttttg cacgtccaat taggtcatta tacacagaat tataccgttg gtgggtcctt 180 cgtatcatac ttatttgccg aatcaagtca aattgatacc ggctctaaag acgtattcca 240 tgtcatagat tggaaatcaa tctttcaatt tcccaaaaga tatgtcacat ttaaaggaaa 300 taatggaaaa tatttagggg ttatcacaat taatcaactt ccatgtctac aatttgggta 360 tgataatctt aatgatccaa aggtggctca tgaaatgttt gtcacttcta atggtactat 420 ttgcattaaa tccacttata tgaacaagtt ttggagactc tctacggatg attggatatt 480 agtcgatggg aatgatcctc gcgaaactaa tgaagctgca gcgttgttta ggtcagatgt 540 gcatgatttt aatgtgattt cgcttttgaa catgcaaaaa acttggttta ttaagagatt 600 tacgagtggt aagcctgggt ttataaattg tatgaatgca gctactcaaa atgttgatga 660 aactgctatt ttagagataa tagaattggg atccaacaac 700 4 304 PRT Amaranthus caudatus amino acid sequence deduced from the two exons (1)..(304) 4 Met Ala Gly Leu Pro Val Ile Met Cys Leu Lys Ser Asn Asn Asn Gln 1 5 10 15 Lys Tyr Leu Arg Tyr Gln Ser Asp Asn Ile Gln Gln Tyr Gly Leu Leu 20 25 30 Gln Phe Ser Ala Asp Lys Ile Leu Asp Pro Leu Ala Gln Phe Glu Val 35 40 45 Glu Pro Ser Lys Thr Tyr Asp Gly Leu Val His Ile Lys Ser Arg Tyr 50 55 60 Thr Asn Lys Tyr Leu Val Arg Trp Ser Pro Asn His Tyr Trp Ile Thr 65 70 75 80 Ala Ser Ala Asn Glu Pro Asp Glu Asn Lys Ser Asn Trp Ala Cys Thr 85 90 95 Leu Phe Lys Pro Leu Tyr Val Glu Glu Gly Asn Met Lys Lys Val Arg 100 105 110 Leu Leu His Val Gln Leu Gly His Tyr Thr Gln Asn Tyr Thr Val Gly 115 120 125 Gly Ser Phe Val Ser Tyr Leu Phe Ala Glu Ser Ser Gln Ile Asp Thr 130 135 140 Gly Ser Lys Asp Val Phe His Val Ile Asp Trp Lys Ser Ile Phe Gln 145 150 155 160 Phe Pro Lys Arg Tyr Val Thr Phe Lys Gly Asn Asn Gly Lys Tyr Leu 165 170 175 Gly Val Ile Thr Ile Asn Gln Leu Pro Cys Leu Gln Phe Gly Tyr Asp 180 185 190 Asn Leu Asn Asp Pro Lys Val Ala His Glu Met Phe Val Thr Ser Asn 195 200 205 Gly Thr Ile Cys Ile Lys Ser Thr Tyr Met Asn Lys Phe Trp Arg Leu 210 215 220 Ser Thr Asp Asp Trp Ile Leu Val Asp Gly Asn Asp Pro Arg Glu Thr 225 230 235 240 Asn Glu Ala Ala Ala Leu Phe Arg Ser Asp Val His Asp Phe Asn Val 245 250 255 Ile Ser Leu Leu Asn Met Gln Lys Thr Trp Phe Ile Lys Arg Phe Thr 260 265 270 Ser Gly Lys Pro Gly Phe Ile Asn Cys Met Asn Ala Ala Thr Gln Asn 275 280 285 Val Asp Glu Thr Ala Ile Leu Glu Ile Ile Glu Leu Gly Ser Asn Asn 290 295 300 

1. A structural gene of Amaranthus caudatus agglutinin (ACA), characterized by a intron and two exons. The gene has a nucleotide sequence of 2628 bp as shown bellow:    1 ATGGCGGGATTACCAGTGATTATGTGCCTAAAATCAAATAACAACCAG AAGTACTTAAGATATCAAAGTGATAATATTCAACAATATGGTCTTCTT CAATTTTGAGCTGATAAGATTTTAGATCCATTAGCTCAATTTGAAGTC GAACCTTCCAAGACTTATGATGGTCTTGTTCACATCAAATCTCGCTAC ACTAACAAATATTTGGTTAGgtacgttttttttcgcgtttatacttcc tccgtttcaatatagtcgcaatatatcgaaaataaacactattcactt atctcctttaatttgtgattgataagtaatttataagttaaaacatag tcaactgagatcttgtttgattcatctcgacgtaaagattattaatat caaatttttataattttatattatacataattgtagttattaatgatt gaattagtacattagactgcgtgaaaaaggcatatgttgtaagtattt tggaacgaaggtagtgttatatttcttctgttagtctatttaaaattt gcgatatttaaattaaatatttaagatttttttgagttacatgtttca aattctggagtgagtacgtactttaagtatacttttgtactagctacc atttgattgacataattaataaatgggaaattccacatggtagcatct agttttgatgaaatgccagtggtgacacttttttaagaaaattccaca tgctagcattcagtttatacactatctaattgccgtagcattttccgt taagttcttgttaaattccactaaatttatcattttgtaagttttttt ccacatggtagtatccagtttttgttaatttatcattttaactcttaa ttcaccattttaatgtttaattcactatttaattcattaacgctcata agtgttagatatttttattgaaaaattataagaaactatctaatcggg tcgaaacaagaacttattcgcctatttttcgatacgtaaaccggaaaa gatatttggcgtcatttttacctatcataacgatttttttcaataaac ggacgttgcaattacccttatgggataactctttcgaaaatccggtcg aaacaagtacttattcgcctattttccgatagatgtgttcaagaaaat gggacaactaaatagattcagagggagtatttcggagtaattatgttg acaaaattatacttgaactaatagctacggtgatgcttgttcaatttc actgattttgtacaatcataaagtttcttgatcacatttccaaaaaaa tgaaagttaagtggcaaaaaatatgtgaatataaaatttgacaagtct aatttaaaattttcactaattttttttaataaaacgaaatacaacata atatatttttattgagatatattttgtgaaatttaatttaaatgtcac cactataaatttgtaatggtacatatagttgatttagttacatttatt agaccttctagctgcataattaaaatcatactaaagctttttcctgag atagaatctaatgatatttctcatatgccggcatgcaaccaaataaag tgttactatataaattactaaagtagcgacattttttaatgttgcaaa aagaaaattttgtttagtgacggtcttatagtgtgactatctctattg ggcagacatatgtacatttagtatgaaatgatcacttataattttaaa gtaatacaattacagagtgacttgtttacaatgaatttgtgtttttgt ccagcaagtcttttaatgagagacgtctcttagatctaatccattaga gtttaattcttactcttattctatatttttctttatgggtcacccaat tagagttggtatctcaaagagaccgtctctcacaagaatttgcgtttt tgtcttagGTGGTCTCCCAATCATTATTGGATTACAGCATCAGCCAAT GAACCAGATGAAAATAAAAGCAATTGGGCATGCACATTATTCAAACCA CTTTACGTAGAAGAAGGTAACATGAAAAAGGTTCGACTTTTGCACGTC CAATTAGGTCATTATACACAGAATTATACCGTTGGTGGGTCCTTCGTA TCATACTTATTTGCCGAATCAAGTCAAATTGATACCCGGTCTAAAGAC GTATTCCATGTCATAGATTGGAAATCAATCTTTCAATTTCCCAAAAGA TATGTCAGATTTAAAGGAAATAATGGAAAATATTTAGGGGTTATCACA ATTAATCAACTTCCATGTCTACAATTTGGGTATGATAATGTTAATGAT CCAAAGGTGGCTCATGAAATGTTTGTCACTTCTAATGGTACTATTTGC ATTAAATCCACTTATATGAACAAGTTTTGGAGACTCTCTACGGATGAT TGGATATTAGTCGATGGGAATGATCCTCGCGAAACTAATGAAGCTGCA GCGTTGTTTAGGTCAGATGTGCATGATTTTAATGTGATTTCGCTTTTG AACATGCAAAAAACTTGGTTTATTAAGAGATTTACGAGTGGTAAGCCT GGGTTTATAAATTGTATGAATGCAGCTACTCAAAATGTTGATGAAACT                                   2628    GCTATTTTAGAGATAATAGAATTGGGATCCAACAAC


2. The two exons—exon 1 and exon 2 according to claim 1, comprising nucleotide sequences of 212 bp and 700 bp respectively as shown below: Exon 1: ATGGCGGGATTACCAGTGATTATGTGCCTAAAATCAAATAACAACCAG AAGTACTTAAGATATCAAAGTGATAATATTCAACAATATGGTCTTCTT CAATTTTCAGCTGATAAGATTTTAGATCCATTAGCTCAATTTGAAGTC GAACCTTCCAAGACTTATGATGGTCTTGTTCACATCAAATCTCGCTAC                       212    ACTAACAAATATTTGGTTAG Exon 2    1929 GTGGTCTCCCAATCATTATTGGATTACAGCATCAGCCAATGAAGCAGA TGAAAATAAAAGCAATTGGGCATGCAGATTATTCAAACCACTTTACGT AGAAGAAGGTAACATGAAAAAGGTTCGACTTTTGCACGTCCAATTAGG TCATTATACACAGAATTATACCGTTGGTGGGTCCTTCGTATCATACTT ATTTGCCGAATCAAGTCAAATTGATACCGGCTCTAAAGACGTATTCCA TGTCATAGATTGGAAATCAATCTTTCAATTTCCCAAAAGATATGTCAC ATTTAAAGGAAATAATGGAAAATATTTAGGGGTTATCACAATTAATCA ACTTCCATGTGTACAATTTGGGTATGATAATCTTAATGATCCAAAGGT GGCTCATGAAATGTTTGTCACTTCTAATGGTACTATTTGCATTAAATC CACTTATATGAACAAGTTTTGGAGACTCTCTACGGATGATTGGATATT AGTCGATGGGAATGATCCTCGCGAAACTAATGAAGCTGCAGCGTTGTT TAGGTCAGATGTGCATGATTTTAATGTGATTTCGCTTTTGAACATGCA AAAAACTTGGTTTATTAAGAGATTTACGAGTGGTAAGCCTGGGTTTAT AAATTGTATGAATGCAGCTACTCAAAATGTTGATGAAACTGCTATTTT                           2628    AGAGATAATAGAATTGGGATCCAACAAC


3. A amino acid sequence deduced from the said two exons according to claim 2 is presented bellow: 1 M A G L P V I M C L K S N N N Q K Y L R Y Q S D N I Q Q Y G L L Q F S A D K I L D P L A Q F E V E P S K T Y D G L V H I K S R Y T N K Y L V R W S P N H Y W I T A S A N E P D E N K S N W A C T L F K P L Y V E E G N M K K V R L L H V Q L G H Y T Q N Y T V G G S F V S Y L F A E S S Q I D T G S K D V F H V I D W K S I F Q F P K R Y V T F K G N N G K Y L G V I T I N Q L P C L Q F G Y D N L N D P K V A H E M F V T S N G T I C I K S T Y M N K F W R L S T D D W I L V D G N D P R E T N E A A A L F R S D V H D F N V I S L L N M Q K T W F I K R F T S G K P G F I N C M N A A T Q N V D E T A I L E I I E L       304 G S N N .


4. A recombinant plasmid, comprising a pACA plasmid having the said ACA gene sequence of claim
 1. 5. A recombinant plasmid, comprising pACAc plasmid which contains only the exons sequences of the ACA gene as described in claim
 2. 6. A recombinant microbe, comprising the recombinant plasmid pACA or pACAc as described in claim 4 or
 5. 7. The recombinant microbe according to claim 6, it is Escherichia coli DH5α, with the accession No. CGMCC No.
 0438. 8. A recombinant plasmid, it is a plant expression vector constructed using the ACA gene sequence described in claim 1 or 2 and a binary expression vector pBin438.
 9. The plant expression vector according to claim 8, they are pBACA and pBACAc shown in FIG. 5 or 6 respectively.
 10. A recombinant plasmid, it is constructed using the exons sequences of the ACA gene described in claim 2 and a E. coli expression vector pET30a (+).
 11. The recombinant plasmid according to claim 10 is pEACAc.
 12. A recombinant microbe, comprising the recombinant pasmid pBACA or the recombinant plasmid pBACAc.
 13. A recombinant microbe, comprising the recombinant plasmid pEACA.
 14. The recombinant microbe according to claim 12 is Agrobacterium tumefacieus LBA4404 or Escherichia coli DH5α.
 15. An application of the plant expression vector pBACA or pBACAc described in claim 9 in transformation of plants to obtain transgenic plants.
 16. An application of the E. coli expression vector pEACAc comprising the sequence of the ACA gene coding region in expressing ACA agglutinin protein in bacteria. 